For the control of the metabolism of diabetic patients the determination of glycosylated hemoglobins in diabetic surveillance has proved to be an informative and reliable process in clinical practice, in addition to the determination of blood and urine sugars.
Hemoglobin, the pigment of the blood corpuscles, consists of approximately 5% of a coloring component, the heme, and approximately 95% of a protein, the globin, which are bonded together in the manner of a complex.
It has now been found that in the column chromatographic separation of hemoglobin solutions the principal hemoglobin portion, namely HbA.sub.I, is inhomogeneous, i.e., in front of the main fraction a series of more rapidly migrating hemoglobins, designated HbA.sub.1, were eluted. These hemoglobin fractions may be separated into the components HbA.sub.Ia, HbA.sub.Ib and HbA.sub.Ic. Chemical structure determinations showed that glycoproteins, i.e., glycosylation products of hemoglobin A.sub.I are involved, formed by a condensation reaction of tne aldehyde group of carbohydrate molecules, such as fructose-1,6-diphosphate, glucose-6-phosphate and glucose with terminal amino groups. An equilibrium reaction initially leads to the formation of the unstable aldimine (Schiff's base), which by way of an amadorium orientation leads irreversibly to the ketoamine. The glycosylated hemoglobin (HbA.sub.Ia, HbA.sub.Ib and HbA.sub.Ic) is formed in a non-enzymatic reaction during the life of the erythrocytes of approximately 120 days.
While in persons without diabetes and with a healthy metabolism and proportion of glycosylated hemoglobin is between 3.5 and 7.5%, in the case of diabetic patients it may be increased to 20% and more, with respect to the total Hb.
U.S. Pat. No. 3,964,865 discloses a lyophilized hemoglobin standard for the colorimetric determination of total hemoglobin, prepared by filtering a "crude" hemoglobin fraction wherein by means of the atmospheric oxygen introduced by the filtration, the hemoglobin is oxidized to methemoglobin, i.e., to oxyhemoglobin. After adjustment to a predetermined concentration value, the methemoglobin is lyophilized and is suitable in this form as a standard agent for the colorimetric determination of total hemoglobin. In order to comply with the standards established by the National Academy of Sciences--National Research Council, USA (NRC) and the International Committee for Standardization in Haematology (ICSH), the lyophilized standard is converted to cyanmethemoglobin prior to use.
However, with this state of the art standard, only a spectroscopic determination of the total hemoglobin is possible, but not a determination of individual hemoglobin fractions, such as for example the HbA.sub.I fraction, since in the oxidation of hemoglobin to methemoglobin both the prosthetic groups and the orientation behavior of the chains are altered, so that such a standard exhibits for example a chromatographic behavior other than that of untreated blood and therefore cannot be used either for a direct determination of hemoglobin fractions nor as a standard for the correct handling of the separation process chosen for example for the determination of HbA.sub.I.
For the determination of the proportion of glycosylated hemoglobin in the total hemoglobin, different processes are available. In view of the stronger negative charge of glycosylated hemoglobins HbA.sub.I, the latter may be separated from other hemoglobins by means of ion exchange chromatography, high pressure liquid chromatography and electro-osmosis. A further method for the determination of glycosylated hemoglobins is based on the hydrolysis of the glycosylated hemoglobins in oxalic acid and conversion of the hexoses split off to 5-hydroxymethylfurfural, which after reaction with thiobarbituric acid (TBA) may be measured photometrically. An affinity chromatographic separation, using the hydroxyl groups of the carbohydrates bound to the hemoglobin, is also possible.
The use of the different methods of determination leads, however, to the disadvantage that the analytical results obtained with the same sample correlate differently with each other, depending on the method applied. Thus, for example, regardless of whether macro- or microcolumns are used for the proportional content of glycohemoglobin, the HbA.sub.I values found by the chemical TBA method are comparable by means of a correction factor only. Furthermore, in the usual analytical processes in part the total HbA.sub.I fraction and the separated individual HbA.sub.Ia,b and HbA.sub.Ic fractions are determined, so that analytical values cannot be given uniformly and are therefore difficult to compare with each other. If, for example, the glycosylated hemoglobin components HbA.sub.Ia+b+c, which are readily determined both by conventional macrocolumn chromatography and by high pressure liquid chromatography, are analyzed, the evaluation of the results merely shows a correlation but no agreement between the two methods.
In summary, it may be stated that the individual methods for the determination of glycosylated hemoglobins, in particular ion exchange chromatography, yield good results with respect to the separation of the fractions, but that these results are not, depending on the process used, transferable and thus comparable. For the internal control of specific separation methods at the present time standard controls are used, which are applicable to a certain process only. However, the comparability of all of the separation techniques applied and thus of the values found by means of a universal standard remains an important requirement of routine clinical examinations.
It is the object of the present invention to provide a process for the preparation of a stable glycosylated hemoglobin standard to make possible the quality control of the HbA.sub.I determination by all of the separation method used; both internally and externally with an identical material.
This object is attained in the process according to the present invention.